The therapy of AIDS has expanded over the years to include 26 drugs, including the latest drug Fuzeon, a fusion inhibitor, in addition to the longer developed HIV reverse transcriptase and protease inhibitor agents (RTI and PI, respectively). However, rapid development of drug resistant viral strains makes mono- and bi- therapy for HIV infection only transiently effective. In addition, Fuzeon cannot be used orally, and the administration of combination RTI and PI drugs requires complicated dosing schedules, which are hard to maintain, and is quite costly. Therefore, continued effort for the discovery of new anti-HIV drugs, especially for new small molecules with novel anti-HIV mechanisms, is still needed. In the course of our discovery and development of Bevirimat, a modified triterpene and the first-in-class HIV maturation inhibitor, we have also discovered that appropriately substituted triterpenes show potent HIV entry inhibition. Thus, the overall goal of this research is to further study modified triterpenes as potent HIV-1 entry inhibitors. We hypothesize that new triterpenes with a pharmacological profile superior to current leads can be identified through structural optimization of current leads or new triterpenes from natural sources. This project will be carried out as a collaborative interdisciplinary effort to enhance the translational research for discovering novel potent anti-AIDS drugs. The hypothesis will be tested and the goal will be achieved through the following specific aims. 1. To discover new natural product-derived triterpenes/leads that can inhibit HIV-1 entry. Selection of triterpenes will be based upon their demonstrated anti-HIV activity profiles. 2. To obtain a next generation of triterpenes with improved anti-HIV-1 entry activity through lead optimization. Extensive modification of betulinic acid will be performed to produce the best anti-HIV entry inhibitors. The modification will focus on the C-28 side chain, C-19 isopropenyl moiety, and ring-A. The structures conferring the best anti-HIV entry activity for betulinic acid will be applied to the modification of promising leads identified from the studies described in Specific Aim 1. All natural and modified triterpenes will be bioassayed for their inhibition of HIV replication and entry. The compounds with superior anti-HIV-1 entry activity to our current leads will be further tested against a panel of genetically diverse HIV-1 strains, including drug-resistant viruses. 3. To determine the molecular mechanism of action. The objective of this aim is to identify the drug binding site of the triterpenes. Information about the drug binding site is critical for further drug design and development, including structural optimization of the compounds and potential combination with other anti-HIV-1 drugs. Our approach to achieve this aim is to cross-link the active triterpenes to the target viral molecules (gp120) and analyze the amino acid residues that are involved in the drug binding site. With good preliminary data, it is very likely that novel promising compounds will be discovered. It is highly probable that the pre-clinical drug candidates generated from this project can be developed in collaboration with a pharmaceutical company to enter clinical trials, and possibly succeed later on as new anti-HIV drugs targeting viral entry. PUBLIC HEALTH RELEVANCE: Based on good preliminary data and an identified lead compound, this research intends an extensive study to produce an optimal modified triterpene that will inhibit HIV-1 entry. This viral stage is currently targeted only by the drug, Fuzeon, which has drawbacks of non-oral administration and high cost. Structural modification of multiple triterpene skeletons and mechanism of action studies are planned in this study to develop an orally bioavailable compound in a cost-effective manner. The aim is to discover an optimal compound that, with the help of a pharmaceutical partner, can be developed as an anti- AIDS clinical trial candidate and, ultimately, as a new drug to treat AIDS-infected patients.